Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Summary

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Abstract

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

Introduction

Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages.

Tissue macrophages may be either resident within tissue....

Protocol

Leukapheresis was carried out under a human subject's research protocol approved by a National Institutes of Health institutional review board.

1. Isolation and Cryopreservation of Monocytes

1. Obtain mononuclear cells by leukapheresis of human donors, and enrich monocytes by continuous counter-flow centrifugation elutriation of mononuclear cells as described in the references ^7,8. Obtain approximately 100 x 10⁶ elutriated cells (approximately 80 - 90% monocytes) in elutriation buffer specified by the manufacturer. Count cells using a hemocytometer.
2. Centrifuge monocytes in a 15 ml polypropylene t....

Results

The viability of fresh or cryopreserved monocytes was greater than 95% as determined with Trypan Blue staining ⁹. Figure 1 and Figure 2 compare at lower and higher magnifications, respectively, the progress of fresh and cryopreserved (i.e., frozen) monocyte differentiation into macrophages. Note that the fresh compared with cryopreserved monocytes show a subpopulation of differentiating monocytes that do not spread but.......

Discussion

Generating defined macrophage types can clarify some of the conflicting results obtained by investigators when studying macrophage biology. The use of various culture conditions and differentiation factors to generate primary human macrophages can lead to very different macrophage types, a fact that may not be appreciated by the researcher. For example, macrophages sometimes are generated from human monocytes using no serum, human serum alone, human serum supplemented with M-CSF, FBS alone, or FBS containing M-CSF or GM-.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cellbind 12-well culture plate	Corning	3336
CELLSTAR, T-75 flask, tissue culture treated	Greiner Bio-One North America	658157
RPMI 1640 culture medium	Cellgro Mediatech	15-040-CM	warmed to 37 °C
L-Glutamine	Cellgro Mediatech	25-005-CI
Fetal bovine serum	Gibco	16000-036
M-CSF	PeproTech	300-25
GM-CSF	PeproTech	300-03
IL-10	PeproTech	200-10
DMSO	Sigma	D2650
Cryovial	Thermo Scientific	375418
DPBS without Ca2+ and Mg2+	Corning cellgro	21-031-CV
0.25% Trypsin-EDTA	Gibco	25200-056
50 ml polypropylene conical tube	Falcon	352070
Trypan Blue	Lonza	17-942E
Neubauer-improved bright light hemocytometer	Paul Marienfeld GmbH & Co. KG	610031	http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html
CoolCell LX cell freezing container	BioCision	BCS-405	other freezing containers also should  be adequate for this step
Liquid Nitrogen Storage System, CryoPlus 1	Thermo Scientific	7400	any liquid nitrogen tank should be adequate